Attempts to separate the ATPase activity from the bulk of the spectrin will be made, since it is not clear whether the activity is due to a small fraction of contaminant peptides ("erythrocytic myosins") or to a low level, widely distributed activity, or to a kinase-phosphatase couple. To date fractionation with deoxycholate chromatography or Triton extraction has been unsuccessful, in that the activity has simply vanished. Further attempts will be made with guanidine chromatography, actin affinity chromatography, and isoelectric focussing. Studies of spectrin and muscle actin will continue, with emphasis on the interactions found in the presence of physiological concentrations of KCl, Mg, ATP and trace calcium. These studies will be made in parallel with spin labelling and by electron microscopy. We will attempt reconstitution of spectrin-lipid complexes by binding lipid to spectrin-actin networks derived from Triton-extracted cells or ghosts. We will attempt, by mixing networks with Triton-solubilized lipids and diluting followed by column chromatography and/or centrifugation, to remove Triton. Assay tools will be electron microscopy, lipid and protein determination, and spin labelling, where the label will be attached to networks before extraction, or will be included in the lipid before reconstitution. We plan to measure both the radioactive and cold phosphate contents of spectrin as a function of time of incubation at 37 degrees in medium with and without glucose, to evaluate the rate of turnover of spectrin phosphate. Spectrin will be isolated by Triton extraction, freed of nucleotides (by TCA) and lipids (chloroform-methanol) and counted (for total P-32), run on gels and autoradiographed (for location of P-32) and dry-ashed and assayed for chemical phosphate. BIBLIOGRAPHIC REFERENCES: Kirkpatrick, F.H. Spectrin: Current Understanding of its Physical, Biochemical, and Functional Properties. Life Sci. 19:1, 1976. Kirkpatrick, F.H., G.M. Woods, R.I. Weed and P.L. La Celle. Fractionation of Spectrin by Differential Precipitation with Calcium. Arch. Biochem. Biophys. 175:367, 1976.